Inhibitory action of oxaloacetate on succinate oxidation in rat-liver mitochondria and the mechanism of its reversal

Inhibitory action of oxaloacetate on succinate oxidation in rat-liver mitochondria and the mechanism of its reversal


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I N H I B I T O R Y ACTION OF OXALOACETATE ON SUCCINATE OXIDATION IN RAT-LIVER MITOCHONDRIA AND T H E MECHANISM OF ITS REVERSAL ANNA B. \VOJTCZAK Depart~nent of Biochemistry, Nencki Institute of Experime~tal Biology, Warsaw 22 (Poland) (Received May z4th , I968)

SUMMARY x. Oxidation of succinate in rat-liver mitochondria was studied in the presence of added oxaloacetate, an uncoupler of oxidative phosphorylation and rotenone. z. At certain concentrations of oxaloacetate, succinate and nfitochondria a spontaneous reactivation of succinate oxidation, previously inhibited by oxaloacetate, can be observed. This reactivation is completely abolished by either arsenate, arsenite or lewisite or by the absence of phosphate, and is potentiated by oligontycin. The spontaneous reactivation is accompanied by an increase in the intranfitochondrial content of ATP generated by substrate-level phosphorylation. 3. ATP added externally reactivates the oxidation of succinate substantially only in the presence of either carnitine or, to a smaller degree, arsenate. 4. The reactivation produced by ATP plus carnitine is potentiated by the addition of small amounts of palmitate. The addition of pyruvate has a small effect and the addition of pyruvate p!us arsenate almost none. 5. Freshly isolated rat-liver nfitochondria contain I5-3o m/mloles free fatty acids per mg protein. This amount is increased during incubation in the presence of KCN, but (luring aerobic incubation with succinate fatty acids are oxidized, even if 2,4-dinitrophenol is present. 6. Upon addition of oxaloacetate the oxidation of intramitochondrial nicotinanfide nucleotides is observed. The steady-state redox level depends on the concentration of added oxaloacetate and on the presence of oxidizable NAD-linked substrates. In the presence of oxaloacetate, intramitochondrial NAD(P) + is most effectively reduced by isocitrate ; ATP phts carnitine having a small effect only. 7. The best protection of succinate oxidation against the inhibition by oxaloacetate is provided by ATP plus carnitine, by ATP plus carnitine plus palnlitate, or by pahuitoyl-carnitine. S. It is concluded that the oxidation of fatty acids is the most effective factor renloving oxaloacetate from the site of succinate dehydrogenase in liver mitochondria. Its effect is due to (i) generation of NADH which can reduce oxaloacetate to malate, Abbreviations: CCCP, carbonyl-cyanide m-chlorophenylhydrazone; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; EGTA, ethyleneglycol bis(fl-aminoethyl ether).£:,N'-tctraacetate.

Bioehim. Biophys. Acta, I72 (I969) 52-65



and (ii) provision of acetyl-CoA which can condense with oxaloacetate to form citrate. A smaller effectiveness of other reactions removing oxaloacetate from mitochondria is discussed.

INTRODUCTION Oxaloacetate is a potent inhibitor of succinate dehydrogenase (EC, 2. However, m a n y studies on the effect of oxaloacetate on succinate oxidation in intact mitochondria revealed the complexity of this inhibition. SCHOLLMEYERAND KLINGENBERG3 and KVN# have shown that succinate m a y be oxidized by intact mitochondria under specific conditions even in the presence of relatively high concentrations of oxaloacetate in the medium. On the other hand, AKERBLOMel al. ~ have observed a strong inhibition of succinate oxidation even in cases when the concentration of oxaloacetate was extremely low. These differences m a y be attributed on the one side to different permeability of mitochondria towards oxaloacetate, and on the other side to the removal of oxaloacetate by various metabolic processes occurring in mitochondria. They are: (I) transamination with glutamate; (2) reduction to malate; (3) decarboxylation to pyruvate; (4) decarboxylation to phosphoenolpyruvate; (5) condensation with acetyl-CoA to citrate or to malonyl-CoA and pyruvate. However, under specific conditions only some of these mechanisms can be effective. It is known that succinate oxidation in phosphorylating mitochondria is less sensitive to oxaloacetate than under uncoupled conditions6, ~. This is an indication that some of the processes removing oxaloacetate are energy-dependent. The present paper describes the effect of added oxaloacetate on the oxidation of succinate in rat-liver mitochondria. The endogenous formation of oxaloacetate is prevented by rotenone, and the main source oI energy, i.e. respiratory chain-linked oxidative phosphorylation, is blocked by an uncoupler. It is shown that under these conditions succinate oxidation can be protected against oxaloacetate inhibition b y ATP formed in substrate-level phosphorylation or externally added. This ATP (or GTP) is needed for the activation of endogenous fatty acids whose oxidation provides reducing equivalents and acetyl-CoA, both effective in removing oxaloacetate from mitochondria. It is thus suggested that in rat-liver mitochondria the main factors controlling the level of oxaloacetate are the reduction to malate and the condensation to citrate. METHODS Rat-liver mitochondria were isolated according to HOGEBOOM8 in o.25 M sucrose + I mM Tris-HC1 (pH 7.4)- The isolation medium for mitochondria used in swellingcontraction experiments also contained o.I mM EDTA. 02 uptake was measured with Clark type oxygen electrode. The simultaneous recording of light scattering and 02 uptake was accomplished by a device described by CHAPPELL AND CROFTS9. ATP was determined enzymatically with hexokinase (EC 2.7.1.i) and glucose-6phosphate dehydrogenase (EC l°. Fluorescence was measured in Eppendorf fluorimeter. Biochim. Biophys. Acta, 172

(1969) 52-65



Free fatty acids were determined by microtitration according to DOLE AND MEINERTZn. The extraction and separation of free fatty acids were as follows. Incubation samples were inactivated by immersing for 2-3 min in a water bath at IOO° and were centrifuged. The precipitated protein was extracted several times with a mixture of ethyl ether and methanol (2:I, v/v) at 3 o°. Conlbined extracts were evaporated under N 2 and the residue was dissolved in petroleum ether. The ether solution was then shaken with equal volume of cold alkaline ethanol (o.o6 M NaOH in 5o % ethanol) ; the ether layer was separated and extracted twice with new portions of alkaline ethanol, and finally discarded (it contained esterified fatty acids, mainly phospholipids). All extractions with alkaline ethanol were carried out at o ° to prevent the hydrolysis of phospholipids. Combined ethanol layers were acidified with H2SO 4 to pH 3-4 and extracted several times with petroleum ether. Combined petroleum ether extracts were washed with water and used for the titrimetric estimation of fatty acids. Protein was determined by the biuret method vz after solubilizing mitochondria with deoxycholate. Oxaloacetic acid and ATP were obtained from both Sigma (St. Louis, Mo., U.S.A.) and Boehringer (Mannheim, Germany) and DL-carnitme-HC1 from Light (Colnbrook, Great Britain), hexokinase and glucose-6-phosphate dehydrogenase were from Boehringer. Sodium succinate and other substrates were of analytical grade. DL-Palmitoyl-carnitine was kindly offered by Dr. P. B. GARLAIX'Dand carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl-cynanide mchlorophenylhydrazone (CCCP) by Dr. P. G. HEYTLER of E.I. Du Pont de Nemours and Co. Inc. (Wilnlington, Del., U.S.A.). RESULTS

Permeability of mitochondria to oxaloacetate CHAPPELL AND HAARHOFF13 have shown that various anions differ in their ability to penetrate the mitoehondrial membranes. In this respect the anions can be divided into four groups: (I) freely penetrating; (2) penetrating in the presence of P1; (3) penetrating in the presence of Pi plus L-malate; and (4) non-penetrants. The question thus arose as to the penetrating ability of oxaloacetate. An experinlent carried out in a system described by CHAPPELLAND HAARHOFF13 demonstrated that mitochondrm suspended in o.I M ammonium oxaloacetate did not swell until 2 mM annnonium phosphate was added (Fig. I). This indicates that oxaloacetate belongs to the group of anions, such as succinate, D- and L-malate and malonate, whose penetration through mitochondrial menlbranes is phosphate-dependent.

Effect of oxaloacetate at various energetic s~ales of mitochondria As shown in Fig. 2, the effect of oxaloacetate on succinate oxidation strongly depends on the energetic state of mitochondria. With 3 mM oxaloaeetate no inhibition occurs in States 4 and 3, but the inhibition sets in very quickly when a low energy state is induced by either 2,4-dinitrophenol or gramicidin plus K+. In Trace A the addition of KC1 in the presence of gramicidin induces both an increase of O,, uptake and the swelling of mitochondria. The later effect is a manifestation of an increased transport of ions, i.e. of K + and, most likely, of both succinate and oxaloacetate. The inhibition

Biochim. ]3iophys. Acta, 172 (1969) 52-65



of respiration which appears subsequently might be a result of either an increased penetration of oxaloacetate, or the lowering of the energy state of mitochondria. Traces C and D point to the later possibility. The time course of succi~zate oxidation i~ the presence of oxaloacetate: inhibition and reactivation When various amounts of oxaloacetate were added to the system containing niitochondria (6- 9 mg protein), succinate (5 niM), 2,4-dinitrophenol and rotenone (in the final volume of 3.0 nil), it could be observed that 1-2 mM oxaloacetate had little or no effect on 02 uptake, while 3-4 mM oxaloacetate produced a substantial but transient inhibition followed by a spontaneous reactivation up to 50 % of the initial activity. When the amount of oxaloacetate was further increased the extent of this reactivation gradually decreased and finally disappeared. A typical picture of M P~




lmin Fig.1

no O A ~ Fig. 2

Fig. I. Swelling of mitochondria in solutions of NH4C1, a m m o n i u m oxaloacetate and a m m o n i u m succinate. I n c u b a t i o n media: Ioo mM solutions of (A) NH4C1, (B) a m m o n i u m oxaloacetate, and (C) a m n m n i u m succinate. I n each case 5 mM Tris HC1 (pH 7.4), o.1 mM ethyleneglycol bis(fl-aminoethyl e t h e r ) N . N ' - t e t r a a c e t a t e (EGTA) and i ffM rotenone were present. 2 mM a m m o n i u m p h o s p h a t e (Pt) was added as indicated. Mitochondria, 2.5 mg protein/ml. Temp. 3 o°. D o w n w a r d deflection of light scattering indicates swelling. Fig. 2. Effect of oxaloacetate on succinate oxidation at various energetic states of mitochondria. I n c u b a t i o n m e d i u m : 2oo mM sucrose, io mM Tris-HC1 (pH 7.4), 2 mM Tris phosphate, 5 mM Tris succinate and i ffM rotenone; mitochondrial protein, 1. 3 m g / m l (in Trace A) and 2.o m g / m l (in Traces B, C and D). Temp. 26 °. Additions: mitochondria (M), o.2 ffg/ml gramicidin, o. 5 mM (Trace A) and 3.3 mM (Traces B, C and D) oxaloacetate (OA), 9 mM KC1, 1. 7 mM ADP, 0.2 mM 2.4-dinitrophenol (DNP) (final concentrations). D o w n w a r d deflection of light scattering (L.S., Trace A) indicates mitochondrial swelling. Oligomycin

ATP* Oligomycln



ATP+A502 *Cornitine

ASi,Or AsO~ , o r Lewisite or absence of Pi


Fig. 3. Succinate oxidation in the presence of oxaloacetate u n d e r various conditions. I n c u b a t i o n m e d i u m : 125 mM KCI, Io mM Tris-HC1, 2 mM Pl, 5 mM sodium succinate, i ffM rotenone and 2.o mg mitochondrial protein/ml, p H 7.4; temp. 26 °. Additions: m i t o c h o n d r i a (M), o.a mM 2,4-dinitrophenol (DNP), 3.3 nlM oxaloacetate (OA), 2 ffg/ml oligomycin, 2 mM ATP, I mM DLcarnitine, 3o ffM palmitate, 2 mM arsenate (As1), 2 mM sodium arsenite (AsO2-), 2 ffg/ml lewisite.

I3iochim. Biophys. Acta, 172 (1969) 52-65



the inhibition produced by 3 mM oxaloacetate is shown in Fig. 3, Curve A. The inhibition appears after a short lag period of about 3o-6o sec and is usually followed by a spontaneous slow reactivation. This reactivation occurs when appropriate prop~rtions between succinate, oxaloacetate and mitochondria are kept. The spontaneous reactivation is greatly enhanced by oligomycin (Fig. 3, Curve B) and is con> pletely abolished by the addition of either 9 mM arsenite, ~ mM arsenate, or z mg/ml lewisite (~-chlorovinylarsenious oxide) (Fig. 3, Curve G). The reactivation is also inhibited when Pi is omitted from the medium. The protective effect of P1 can also be visualized when endogenous Pi is bound as strontium salt within the nfitoehondria. In this case a complete inhibition of succinate oxidation is obtained with even lower concentrations of oxaloacetate, in all subsequent experiments 2 mM Pi was included into the incubation medium. This concentration was found to be optimal, while higher concentrations of Pi, above zo raM, potentiated the inhibitory effect of oxaloacetate. "l'Algl.l: 1 C H A N ( ; 1 ; S OF A ' f ' I ) C O N T E N T IN" R A T - L I V F R M I T O C H O N D R I A I ) [ R I N G S U C C I N A T I £ O X I I ) A T U I N

I n c u b a t i o n m e d i u m as in Fig. 3; 3 m M o x a l o a c e t a t c was a d d e d after I rain of i n c u b a t i o n ; o t h e r a d d i t i o n s as i n d i c a t e d ; t e m p . 37:.

A dditions

A T P (nz/~moles/mg protein) afteJ

FCC 1' (o.5/*5I)


I"CCI ~ (o. 5/tM) i oligonlycin (z/,g/'ml) I;('CI ~ (o. 5 l/M) -- a r s e n i t e (e 1riM) FCCI' (0. 5 HIM) I:CCP (o. 5 HM) !- o l i g o m y c i n (2 i~g/m ) I"(X?I ~ (o. 5/,'M) + o l i g o m y c i n (2/~g/inl)


1.28 i. 4 °

( t o x a l o a c e t a t c ) 1.o 4 ( I o x a l o a c e t a t e ) 1.5 l ( + o x a l o a c e t a t e ) o.oo

t.74 2.40

(i oxaloacetate)1.02 ( ~ oxaIoacetafie) 3.20



On the basis of these observations it may be supposed that ATP (or (ITP) generated by the substrate-level phosphorylation plays a role in the reactivation of suecinate oxidation inhibited by oxaloacetate. In order to check this point, the changes of intramitochondrial ATP were followed during the oxidation of succinate in the presence of an uncoupler (FCCP) and oxaloacetate. It appeared (Table I) that the content of ATP rapidly decreases during the first minute of incubation and decreased only slowly after the addition of oxaloacetate (Expts. i and a, 1st row). Inthe presence of oligomycin the content of ATP also decreased during the first minute, but was increased when oxaloacetate was added (Expt. 2, end row, compare with 3rd row). In the presence of arsenite all lnitochondrial ATP disappeared during a few minutes. This synthesis of ATP was due to substrate-level t)hosphorylation as indicated by its insensitivity to lgCCP and oligomycin and the inhibition by arsenite. These experilnents indicate a parallelism between the rate of succinate oxidation in the presence of oxaloacetate and the content of intralnitochondrial ATP. The reactivaiion by A 2/'1)

The effect ot added ATP on tile inhibition by oxaloacetate of suceinate oxidation has often been studied, ill uncoupled kidney mitochondria ATP reversed the inhibi-Hi,ochim. Bio/,hys. A cla, z7-' (~OOc)) 52 (~5



tion G, whereas in uncoupled rat-liver mitochondria it had only a small effect a,14,15. However, AzzoxE AND ERXSTERT,1G observed that ATP reversed the inhibition in rat-liver mitochondna preincubated in the presence of 2,4-dinitrophenol and arsenate. It was therefore interesting to reinvestigate the effect of ATP in our system. Added ATP had a small and variable effect on the inhibition of succinate oxidation by oxaloacetate. In some experiments it potentiated, in others it partly released, tile inhibition. However, ATP plus carnitine always reversed the inhibition (Fig. 3, Curve C). Carnitine alone, in the absence of ATP, had no effect (not shown). The variable effect of ATP alone probably depended on the content of endogenous fatty acids in nfitochondria, as the addition of a small amount of oleate or pahnitate together with ATP always produced a substantial activation of the respiration. This activation was potentiated by carnitine to such an extent that no inhibition by oxaloacetate could be observed. This effect of carnitine strongly suggests the participation of fatty acids in the reactivating action of ATP. This is further illustrated by an experiment where ATP and carnitine were added to the incubation medium before oxaloacetate. In this case the inhibitory effect of oxaloacetate was largely delayed (Fig. 3, Curve D). If then a mieroquantity of pahnitate was added, O 2 uptake was stimulated. This stimulation was partly due to the oxidation of pahnitate (with oxaloacetate and oxygen as electron acceptors) but, as shown by control experiments without succinate, this amounted to less than Io .... .'o of O 2 uptake observed in Trace D (Fig. 3) after the addition of pahnitate. This indicates that a real reactivation of succinate oxidation occurred. The addition of a new portion of ATP was without effect (not shown). This experiment indicates that endogenous fatty acids are the limiting factor in the reactivating effect of ATP. \Vhen endogenous fatty acids are exhausted, added fatty acids are equally active. Oxaloacetate used in these experiments contained 5--Io % pyruvate, it could be therefore supposed that pyruvate is responsible for the removal of oxaloaeetate from mitochondria and, consequently, for the spontaneous reactivation of succinate oxidation. BRF~IF~R~7 has observed that the oxidation of pyruvate in phosphorylating rat-liver mitochondria is depressed by long-chain acylcarnitines which compete with pyruvate for intramitochondrial CoA. In our hands the utilization of o.3 mM pyruvate in uncoupled liver mitochondria was depressed by o.6-2 mM ATP by about 3o %. This observation nfight eeiplain the inhibitory effect of ATP on the spontaneous reactivation. The question thus arises whether the oxidation of pyruvate was exclusively responsible for the spontaneous reactivation. That it was not is shown by the effect of arsenate which completely prevented the spontaneous reactivation (Yig. 3, Curves E and G), although was without effect on the oxidation of pyruvate. (Arsenate uncouples substrate-level phosphorylation coupled to the oxidation of ~-ketoglutaratelS.) It can be thus concluded that pyruvate (added together with oxaloacetate) is oxidized via the tricarboxylic acid cycle with the generation of :4TP due to substratelevel phosphorylation. This ATP (or GTP) then promotes the oxidation of endogenous fatty acids. This process is most probably responsible for the spontaneous reversal of the inhibition of suceinate oxidation. The oxidation of pyruvate alone, if not accompanied by substrate-level phosphorylation, is not effective in this respect, as shown by the experiment with added arsenate. When the oxidation of pyruvate was inhibited by arsenite, ATP plus carnitine had still an activating, although smaller, effect (Fig. 3, Trace F). Biockim. Biophys. Acta, i72 (~909) 5 z 65



The r e a c t i v a t i o n b y added A T P plus arsenate was more efficient t h a n by A T P alone (Fig. 3, compare Traces C a n d E). This can be explained b y assuming t h a t arsenate increases the breakdown of sueeinyl-CoA liberating CoA which can be utilized in f a t t y acid oxidation. This is also suggested b y experiments in which the oxidation of a4C-labelled oleate was measured in the presence of suceinate, ATP, 2,4-dinitrophenol a n d oligomycin (A. B. WOJTCZAK, u n p u b l i s h e d observations). U n d e r these conditions arsenate increased the disappearance of oleate e-fold wlfile the increase produced b y carnitine was 5-fold. The effect of arsenate is thus similar to that of carnitine, although the mechanisms of b o t h effects are different.

Oxidation o/ e,ldoge~zous fal(v acids The experiments described in the preceding section clearly show t h a t the effect of A T P on the inlfibition of succinate oxidation b y oxaloacetate is in rat-liver mitochondria m a i n l y due to the a c t i v a t i o n of endogenous f a t t y acids. To further test this point the level of f a t t y acids was d e t e r m i n e d in isolated m i t o c h o n d r i a u n d e r various conditions of i n c u b a t i o n . Yreshly isolated m i t o c h o n d r i a c o n t a i n from I5 to 3o m/xmoles non-esterified f a t t y acids per mg m i t o c h o n d r i a protein. This a m o u n t increases by a b o u t 5o % during the i n c u b a t i o n at room t e m p e r a t u r e , especially if the respiratory chain is i n h i b i t e d (Table II). I n the presence of succinate and 2,4-dinitrophenol this increase is not observed, suggesting t h a t an oxidation of f a t t y acids occurs. This is completely abolished b y arsenate b u t not b y arsenate plus ATP. The disappearance of f a t t y acids is most p r o b a b l y due m a i n l y to their oxidatlonlg, '-'°. U n d e r specific conditions, especially in the presence of ~-glycerophosphate, fatty, acids are also incorporated into mitochondrial phospholipids 'z~. This process is, however, nluch slower t h a n the oxidation of f a t t y acids and in the present experiments its p a r t i c i p a t i o n in the removal of endogenous f a t t y acids can be neglected. TABLE 1i THE











Incubation mediuin as in Fig. 3; mitochondrial protein 3o.6 nig; linal vol. 2.o mI; temp. 2o . Additions where indicated: 5 mM succinate, o.I mM 2,4-dinitrophenol, 2 niM KCN, ~ mM ATP, -, mM arsenate. Incubation was carried ont for 3° rain under constant shaking. Fatty acids were extracted and determined as described under METHODS.])eterminations of tree fatty acids were done by Miss E. LAG\VINSKA.

A ddiliolts

Fre{fatly acids (/,moles)

None, sample not incubated None (incubated) KCN Succinate ÷ 2,4-dinitrophenol Succinate + 2,4-dinitrophenol {- ATP Suecinate ? 2,4-dinitrophenol ÷ arsenate Succinate i 2,4-dinitrophenol -- ATP -- arsenate

0.9" 1.3 t T--17 0.90 o.72 ~.82 o.66

I n the presence of rotenone f a t t y acids can be oxidized if oxaloacetate is also present. I n this case oxaloacetate is the electron acceptor. This is illustrated b y Table I I I which shows t h a t in the presence of oxaloacetate the oxidation of oleate, as measured b y the formation of acid-soluble o x i d a t i o n products, is i n d e p e n d e n t of the

iochim. Biophys. Acts, 172 (I969) 52 55



presence of rotenone. However, the production of acetoacetate is much greater in the presence of rotenone than in its absence. This fact provides a further evidence that in the presence of rotenone oxaloacetate is an electron acceptor and is therefore less accessible for citrate synthesis (cf. ref. 23). Thus, the oxidation of endogenous or added fatty acids provides both the reducing equivalents, which can reduce oxaloacetate to malate, and acetyl-CoA which can condense with oxaloaeetate to form citrate. The problem thus arises which of these two processes plays an essential part in removing oxaloacetate from the site of succinate dehydrogenase in liver mitochondria. To elucidate this question the effect of oxaloacetate on the oxidoreduction state of intramitochondrial nicotinamide nucleotides was studied. TABLE lI[ OXIDATION OF [I-14C]OLEATE IN RAT-LIVER MITOCHONDRIA Without rotenone: 125 mM KC1, 2 mM p h o s p h a t e buffer a n d io mM Tris-HC1 buffer (pH 7.4), o.i mM 2,4-dinitrophenol, 1. 3 mM oxaloacetate, io /,M [i-14C]oleate (4ooooo c o u n t s / m i n I~C), 5.25 mg mitochondrial protein; final vol. 3.o ml; temp. 26°; incubation time 5 rain. With rotenone: The same conditions as in above except t h a t 5 mM succinate and i /tM rotenone were added and the concentration of oxaloacetate was increased to 3.3 mM. The samples were deproteinized with HC101 and centrifuged, t h e n KC104 was precipitated and removed. A sample of the supern a t a n t was treated with aniline citrate 2~ and 14CO2 evolved was absorbed in N a O H . Counts corresponding to acetoacetate are the counts of this CO s multiplied b y the factor of 2. Additions

None ATP (2 nlM) ATP (2 raM) + carnitine (I mM)

Oxidation products soluble in HCIO 4 (counts/rain) Without rotenone

With rotenone


A cetoacetate


A eetoacetate

43 °00 41500 168300

19oo 2300 30000

33350 47000 250000

10800 11200 72000

Redox changes in nicotinamide mtcleotides Fig. 4A shows that externally added oxaloacetate can oxidize intramitochondrial nicotinamide nucleotides and that the state of oxidation depends on the concentration of oxaloacetate. The oxidation is very rapid, contrary to the effect of oxaloacetate on succinate oxidation where a lag is observed. At a constant level of oxaloacetate a steady state is obtained, resulting from the oxidation of the nueleotides by oxaloacetate from one side and their reduction by NAD-linked substrates from the other side. The level of NAD + reduction at the steady state depends on the oxidizable substrate. As can be seen in Fig. 4 B isocitrate is more effective in preventing the oxidation of nicotinamide nucleotides than is fi-hydroxybutyrate. ATP alone is without effect but together with carnitine it increases the level of reduction; this effect is, however, small as compared to that of isoeitrate (Fig. 4C). A steady state of the oxidoreduction of mitochondrial nicotinamide nucleotides in the presence of oxaloacetate can also be maintained by endogenous substrates, This is, however, possible only in the coupled state (Fig. 4D). The addition of an uncoupler results in an oxidation of the nucleotides, the rate of this oxidation being further strongly increased by arsenite. This experiment supports the view that the oxidation of endogenous substrates requires energy and that substrate-level phosphorylation (blocked by arsenite) may be one of the energy sources. Biochim. Biophys. Acta, i72 (r969) 52-65




-~l~° ~ ~ x ~ o ;


/NAD(P)* reduced

'= ~

}L,__ r-- 2"---~q E






Fig. 4. Redox changes of intramitochondrial NAD(P)+ in the presence of oxaloacetate under various conditions. Incubation medium: 12o mM KC1, 20 mM Tris-HC1 (pH 7.4), 2 mM PI, 2 mM MgCI,z and 2.0 mg mitochondrial protein/ml. Temp. 23 :~. Additions: 3-3 mM succinate (Succ.), 4 nlM isocitrate (Ic.), 2 mM/~-hydroxybutyrate (/~OH), 1.3 mM OL-carnitine (Carn.), 1.5 inM ATP, I ~Vl rotenone (Rot.), i /~M CCCP, 2 mM arsenite (AsO2-) and oxaloacetate (OA) as indicated.

The reactivation by respiratory substrates The effectiveness of various substances in p r e v e n t i n g the i n h i b i t i o n of succinate oxidation b y oxaloacetate or in reversing this inhibition, is shown in Table IV. It can be seen t h a t A T P alone (plus oligomycin to i n h i b i t the ATPase) has no effect. However, A T P plus carnitine, or A T P plus carnitine plus palmitate, or p a l m i t o y l - c a r n i t i n e can reverse the inhibition. A similar effect is exerted b y isocitrate b u t is partially abolished if either arsenate or arsenite (not shown) are present. /3-Hydroxybutyrate is less effective t h a n isocitrate. P y r u v a t e can effectively p r e v e n t the i n h i b i t i o n if present from the b e g i n n i n g of the i n c u b a t i o n , b u t is not able to release the i n h i b i t i o n if it has already occurred. This is in contrast with p a h n i t o y l - c a r n i t i n e or p a l m i t a t e plus carnitine plus A T P which are effective b o t h when present from the b e g i n n i n g of the i n c u b a t i o n a n d added after the inhibition. The comparison of Table IV and Fig. 4 reveals t h a t there is no close parallelism between the ability of an oxidizable substrate to m a i n t a i n a high level of reduced n i c o t i n a m i d e nucleotides in the presence of oxaloacetate a n d the ability to p r e v e n t or reverse the i n h i b i t o r y effect of oxaloacetate on succinate oxidation. Isocitrate is a potent reducing agent for m i t o c h o n d r i a l NAD + plus N A D P + (Figs. 4 B a n d C), a n d is effective in p r e v e n t i n g or reversing the i n h i b i t i o n of suecinate oxidation produced b y oxaloacetate (Table IV). On the other hand, ATP plus carnitine produce only a slight reduction of m i t o c h o n d r i a l n i c o t i n a m i d e nueleotides (Fig. 4C), b u t are as effective in p r e v e n t i n g the i n h i b i t i o n as is isocitrate (Table IV). It is to be n o t e d t h a t in all cases when a s p o n t a n e o u s or induced r e a c t i v a t i o n of succinate oxidation occurred, no s u b s t a n t i a l decrease in oxaloacetate c o n c e n t r a t i o n in the i n c u b a t i o n m e d i u m could be observed.

Biochim. Biophys. Acta, 172 (1969) 52-65






595 595




630 630 595



f l - H y d r o x y b u t y r a t e (5 raM)












34 °



34 °


17 °
































Palmitoyl-carnitine (3 ° pM) None


ATP (0.6 raM) + carnitine (I raM)

ioo I7°

None P a h n i t a t e (33 HM)


51 None

68 P y r u v a t e (4 nlM)









Isocitrate (i raM)

lsocitrate (i raM)

Carnitine (2 raM)


Subsequent additions

T i m e (rain) after the addition of oxaloacetate

ATP (0.6 raM) + carnitine (I mM)



I s o c i t r a t e (i mM)



Arsenate (2 n i M ) + isocitrate (I raM)



Arsenate (2 raM)



ATP (2 mM) + oligomycin (2/~g/ml)

P y r u v a t e (4 raM) + a r s e n a t e (3 mM)


P y r u v a t e (4 raM)




Rate of O 2 uptake (mpatoms/min)

A T P (2 raM) -5 oligonIycin (2 Etg/ml)

Initial additions

* 0 2 exhausted.





Expt. Protein

I n c u b a t i o n m e d i u m as in Fig. 3 ; 3 m M o x a l o a c e t a t e , o. i m M 2 , 4 - d i n i t r o p h e n o l ; t e m p . 26 °.







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The controlling effect of oxaloacetate in succinate oxidation seems now to be well established, but the control of oxaloacetate level in mitochondria presents several intriguing points. One of them is the permeability of mitochondria to exogenous oxaloacetate. The present investigation suggests that mitochondria are permeable to oxaloacetate. This is indicated by (i) an immediate swelling of mitochondria suspended in IOO mM annnonium oxaloacetate, and (ii) a very rapid oxidation of intramitochondrial NAD(P)H by added oxaloacetate (I-3 raM). At lower concentrations of oxaloacetate (below I nlM) its penetration is probably facilitated by an energydependent cation transport, as recently shown by ROBINSON AND CHAPPELL24 and HASLAM AND KREBS 25,

Another problem represents the energy dependence of succinate oxidation. It is well known that this oxidation in uncoupled mitochondria undergoes a spontaneous inhibition after a short period of initial high rate 4,G,Lls. AzzoxE AND ERNST~:td have suggested that the succinate oxidase system is in some way energy-controlled. However, SLATER AND HULSMANN 26 a n d CHAPPELL6 postulated that oxaloacetate formed endogenously was responsible for the inhibition. It has been also showna,6, la that succinate oxidation is more susceptible to added oxaloacetate in tile uncoupled state than in phosphorylating mitochondria. It has been also observed that addition of ATP reverses the inhibition. The present investigation supports the view expressed earlier by SLATER AND H/]TLSMANN26 that the oxidation of endogenous fatty acids promotes the removal of oxaloacetate from mitochondria and that ATP, either formed in mitochondria or added, is necessary for the activation of these acids. There are two mechanisms by which the oxidation of fatty acids can remove oxaloacetate. The first of them is the reduction of oxaloacetate to malate by NADH formed during the oxidation of fatty acids. This mechanism is especially important in the presence of rotenone or amytai, i.e. when the reoxidation of NADH but the respiratory chain is blocked. In fact, in this case oxaloacetate is the electron acceptor for the oxidation of fatty acids (Table III). That this pathway is effective in removing oxaloacetate from mitochondria is also indicated by the fact that other NAD-linked substrates, especially isocitrate, can also protect against oxaloacetate inhibition. Compatible with this are observations that amytal or rotenone effectively protect against the inhibitory effect of added oxaloacctate ~5. ROB~:RTON2v showed that in a submitochondrial particle system oxaloacetate which had been formed during succinate oxidation could be reduced by NADH generated by the reversal of electron transport. This may be in fact the way in which suceinate oxidation is controlled in coupled nfitochondria. However, in systems containing an uncoupler and/or rotenone, like in the present investigation, this mechanism does not occur. In this case NAD ~ is reduced only by endogenous or added NAD-linked substrates. The effectiveness of various substrates depends on several factors, as the redox potential, activities of particular dehydrogenases and the permeability of mitochondriaI melnbranes. It has been shown recently2S, ''~ that the oxidation of anionic substrates can be limited by the rate of their uptake by mitochondria from the medium and that this process is energy-dependent. This observation may explain the fact that pyruvate is less effective in reactivating suceinate oxidati(m when added to uncoupled mitoehondria than is palmitoyl-carnitipe or pahnitate phts carnitine phts ATP whose Biochim. l?,iophys..dora, ~7" (Ic)69) 52-65



penetration through mitochondrial membranes is controlled by a different mechanism and is most probably independent on the supply of energy. The uptake of succinate by mitochondria is also an energy-requiring process and, if the concentration of succinate in the medium is low, it can be a limiting factor in its oxidation 29. This may be another explanation for the energy-dependence of succinate oxidation as postulated by AZZONEAND ERXSTER7. In the present investigation, however, the concentration of succinate was high enough (5 mM) to make its oxidation independent of the energy-requiring uptake. The second mechanism by which oxidation of fatty acids removes oxaloacetate is the generation of acetyl-CoA which condenses with oxaloacetate to form citrate. This mechanism, suggested by SLATER AND H/5'LSMAXX26, should play an important role in removing oxaloacetate, especially in uncoupled state and in the absence of rotenone or amytal, i.e. under conditions when the respiratory chain is maximally active and nicotinamide nucleotides are highly oxidized. It has been observed in the present investigation that in the presence of rotenone and an uncoupler, a complete inhibition of succinate oxidation produced by oxaloacetate is usually followed by a spontaneous reactivation. It is shown that substratelevel phosphorylation plays an important part in this reactivation. Here again the energy is needed for the activation of endogenous fatty acids. In the spontaneous reactivation a certain role is also presumably played by pyruvate which is present, as an impurity, in oxaloacetate preparations and which is also formed from oxaloacetate by its decarboxylation during the incubation. Although pyruvate oxidation itself is not very effective in removing oxaloacetate (Table IV), it initiates the tri-. carboxylic acid cycle and, consequently, gives rise to the generation of GTP in the substrate-level phosphorylation. We have also noticed that arsenate potentiates the effect of ATP, although to a lower extent than carnitine. Arsenate has probably a sparing effect on mitochondrial CoA by releasing it from succinyl-CoA and thus stimulating the oxidation of fatty acids. Most of the authors who studied the effect of ATP on the reversal of oxaloacetate inhibition used nfitochondria pretreated with an uncoupler (2,4-dinitrophenol or dicumarol) p l u s arsenate6,7, ac and therefore the effect of arsenate escaped their attention. The othersa, 14 noticed, however, that ATP alone had little effect on the reversal of the spontaneous inhibition of succinate oxidation in the presence of 2,4-dinitrophenol. In a previous paper ~5 a reactivating effect of serum albumin, if present together with ADP and Pi, was described. It was suggested that albumin binds an inhibitor of succinate oxidation other than oxaloacetate. At present, we rather postulate that the action of serum albumin can be explained by the stimulation of fatty acid oxidation. Besides~ small amounts of fatty acids are introduced into the reaction medium in a form bound to the albumin, and this again could have a beneficial effect. Finally, serum albumin can bind 2,4-dinitrophenol (ref. 31) and re-couple the phosphorylation of added ADP to form ATP. This seems to be the most satisfactory explanation of the observed effect of serum albumin. The rate and the extent of the reactivation depends on the content of the endogenous fattyacids. Undercertain conditions (Fig. 3D) an addition of a small amount of fatty acids is necessary to produce a reactivation. AKERBLOMgt al. 5 have shown that suceinate oxidation in rat-liver mitochondria depleted of endogenous NAD + is very Biochim. Biophys. Acta, 172 (I969) 52-65,



susceptible to low concentrations of oxaloacetate. The inhibition can be, however, abolished by the addition of ATP plus NAD + plus fatty acid, i.e. under conditions when the oxidation of fatty acids is favoured. The authors suggest that an oxidized derivative of fatty acids can regulate the sensitivity of succinate dehydrogenase towards oxaloacetate. However, these results can also be explained in the sense that the oxidation of fatty acids can effectively remove oxaloacetate from inside of the mitochondria, as postulated in the present paper. The present investigation also provides an explanation of cyclic fluctuations in the long-term oxidation of succinate in rat-liver mitochondria a2. An increase in the oxidation rate can be interpreted as the effect of the oxidation of endogenous substrates, mostly fatty acids, on the removal of oxaloacetate. However, when the oxidation of succinate proceeds at maximal rate, the endogenous formation of oxaloacetate exceeds the rate of its removal and the inhibition starts. The accumulation of oxaloacetate enhances the synthesis of citrate and the operation of the tricarboxylic acid cycle which is coupled to the substrate-level phosphorylation, which in turn stimulates the oxidation of new portions of fatty acids. As a result, the oxidation of succinate is restored until an increased production of oxaloacetate again causes an inhibition. The oscillations are most probably based on a high turnover number of succinate dehydrogenase and a great difference between the values of Ki of this enzyme in respect to oxaloacetate (o.2. IO 6--I.5' lO 6 M ; refs. 33, 34), and Km of citrate synthase (EC 4.I.3.7) in respect to oxaloacetate (saturation concentration 4" lO-~ M; ref. 35). The diminution of the amplitude of these cycles is the result of decreasing the amount of endogenous substrates. PAPa, LOFRUMENTO AND QUAGLIARIELLO 36 have postulated that the energydependent decarboxylation of oxaloacetate to phosphoenolpyruvate is a factor controlling succinate oxidation in rabbit-kidney mitochondria. The formation of phosphoenolpyruvate has also been observed in rat-liver mitochondria in long-term experiments by SCHOLTEAND TA6ERaL However, it appears unlikely that in rat-liver nfitochondria ATP promotes the reversal of the inhibitory action of oxaloacetate by this reaction since the phosphoenolpyruvate carboxykinase (EC 4.I.I.3~-) has only a very low activity in these mitochondria. In contrast to kidney, this enzyme in rat liver is mainly located in the cytoplasm aS. In agreement with this, prelinfinary lneasurements carried out in the system used in this investigation, with ATP added, showed a very slow formation of phosphoenolpyruvate as compared to a relativeh- high oxidation of endogenous fatty acids. Nevertheless, in kidney mitochondria which possess much higher activity of phosphoenolpyruvate carboxykinase than rat-liver mitochondria this enzyme may play a substantial role in removing oxaloacetate. In conclusion, the present investigation seems to indicate that the oxidation of fatty acids (endogenous or added) is the main factor controlling the level of oxaloacetate in rat-liver mitochondria and, consequently, the rate of succinate oxidation. However, this conclusion may not apply to mitochondria of liver from other species, like pigeon, rabbit or guinea pig, and of other organs, which differ from rat-liver mitochondria in their enzylne pattern and the content of endogenous substrates. ACKNOWI.EDGEMENTS I wish to thank Dr. J. B. CHaPPELL for his advice and valuable discussions and Prof. P. J. RANDLEfor his hospitality in the Department of Biochemistry, University

Biochhn. 13iophys. Actu, 172 (1,')09) 52-65



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